Chemical Carcinogenesis

Chemical Carcinogenesis Samuel M. Cohen, MD, PhD

University of Nebraska Medical Center Department of Pathology and Microbiology

Omaha, NE

Conflict of Interest Declaration • Consult for Numerous Companies • Research funded by NIH, Private Industry • FEMA Expert Panel

Outline • Basic Principles of Carcinogenesis • Carcinogenic Chemicals • Carcinogenicity Testing • Model of Carcinogenesis • Mode of Action/Human Relevance

History of Chemical Carcinogenesis • John Hill–Cancer of nose and snuff users, 1761. • Sir Percival Pott–Chimney sweeps (scrotal cancer), 1775. • Rehn–Bladder cancer in aniline dye industry, 1895. • Kennaway and Cook–First pure chemical carcinogen,

dibenz(a,h) anthracene, 1930. • Hueper–2-naphthylamine as bladder carcinogen in dogs,

latency in carcinogenesis, 1937. • Miller and Miller–Ultimate carcinogenic metabolites are

electrophiles adducting DNA, 1967.

Cancer Requires Genetic Alterations • Usually occur as somatic alterations • Can occasionally be inherited • Multiple alterations are required

Inherited Diseases with High Tumor Incidence

• Retinoblastoma • Multiple polyposis coli • Thyroid medullary carcinoma • Multiple endocrine adenomas • Von Recklinghausen’s disease

Diseases Associated with Increased Cancer Risk • UV Radiation

– Albinism – Xeroderma pigmentosm

• Chromosome fragility syndrome – Bloom’s syndrome – Fanconi’s syndrome

• Immunodeficiencies – X-linked lymphoproliferative disease (XLP) – Ataxic-telangiectasia – Severe combined immunodeficiency – Wiskott-Aldrich Syndrome

Chronic Myelogenous Leukemia: Philadelphia Chromosome Normal Configuration of Chromosomes 9 & 22

Rearranged Chromosomes 9 (9q+) & 22 (Ph)

WILD TYPE

PREDISPOSED PHENOTYPE

TUMOR

HEREDITARY

NON-HEREDITARY (Sporadic)

Tumor Suppressor Genes

Oncogenes and Suppressor Genes • Growth factors • Growth factor receptors • Cellular growth signal transducers • Nuclear proteins regulating cell division • Nuclear proteins regulating replication

mechanics • Apoptosis regulators

Immunosurveillance and Cancer • Tumor specific transplantation antigens

– Viral specific in mice – No tumor specific antigens in humans

• Carcinogens are immunosuppressive

– Frequently only at doses >>carcinogenic dose

• Increased incidence of tumors in immunodeficient patients – Only a few specific types of cancer

• Neoplastic clone escapes immune surveillance

Epstein Barr Virus (EBV)

Human Papilloma

Virus (HPV)

Human Kaposi

Sarcoma Virus

(HHV8)

Hepatitis B Virus (HBV)

B-Cell Proliferation

B-Cell Lymphoma

Squamous Cell

Dysplasia

Squamous Cell Carcinoma

Chronic Active

Hepatitis

Hepatoma

Immune Surveillance of Infectious Organisms

Immunosuppression

New Infections or Reactivation

Kaposi Sarcoma

Immunosurveillance and Cancer

Oxidation Conjugation

XENOBIOTIC

LIPOPHILIC

EXCRETION

HYDROPHILIC (Ionizable)

Expose or Add

Functional Groups

Oxidation Reduction Hydrolysis

Primary Product

Secondary Product

Biosynthetic

Conjugations

Foreign Compounds

Xenobiotic Metabolism

1) No Tumors

2) No Tumors

3) Many Tumors

4) No Tumors

5) Many Tumors

6) No Tumors

Symbols: Time

Initiator Promoter

Initiation-Promotion

Limitations of Initiation-Promotion Model • Empirically defined – model dependent • Based on short-term studies • Assumes intermediate benign clonal expression

– Many human tumors do not have this • Sequential administration

– Humans frequently exposed to agents concurrently • Terms have become used with a variety of meanings,

rarely specified and usually unclear

Armitage-Doll Multistep Cancer Model

. . .

μ 0

μ 1

μ 2 μ k-1

Tumor

Normal Cell (N)

Stage 1 Cells

Stage 2 Cells

Malignant Cells (M) (Stage K)

Armitage-Doll Multistage Model I(t) = N λ0λ1 … λn-1tn-1 / (n-1)!

I(t) = incidence at time of t

N = number of normal stem cells

λ = rate of transition between stages

n = number of stages

N, λ assumed to be constant

Cell proliferation rates assumed to be constant

Childhood tumors

Testicular Germ cell tumors

Armitage-Doll prediction

Hodgkin’s Disease Tu

mor

inci

denc

e

Age

Age-Related Cancer Incidence

Cancer Arises Due to “Bad Luck” Variation in cancer risk among tissues can be explained by the number of stem cell divisions Cristian Tomasetti and Bert Vogelstein Science 347:78-81, 2015 Substantial contribution of extrinsic risk factors to cancer development Song Wu, Scott Powers, Wei Zhu & Yusuf A. Hannun Nature 529:43-47, 2016

What We Know Genetic alterations required for cancer

formation

More than one genetic alteration required

DNA replication fidelity is not 100%

Means of Increasing Risk of Cancer Increase Rate of DNA Damage Per Cell

Division (DNA Reactive)

Increase Number of Cell Divisions (Non-DNA Reactive; Increased Cell Proliferation)

Cohen, Ellwein, and Greenfield Model Malignant

Differentiated Cell

Malignant Committed

Cell

Intermediate Differentiated

Cell

Intermediate Committed

Cell

Normal Stem Cell

Normal Differentiated

Cell

Normal Committed

Cell

Intermediate Stem Cell

P1

Malignant Stem Cell

P2

Polycyclic Aromatic Hydrocarbons

Anthracene Benz(a)anthracene

3-Methylcholanthrene 7,12-Dimethylbenz(a)anthracene

Benzo(a)pyrene Dibenz(a,h)anthracene

H3C

CH3

CH3

Aromatic and Heterocyclic Aromatic Amines

2-Naphthylamine 1-Naphthylamine

Benzidine (4,4’-diaminobiphenyl)

2-Aminoflourene

6-Aminochrysene NH2

NH2

NH2

NH2

H2 N

o-Toluidine

NH2

OCH3

H3C

p-Cresidine 4-Aminostilbene

NH2

6-Aminochrysene NH2

NH2

OCH3

H3C

p-Cresidine

NHCOCH3

OCH2CH3 Phenacetin

NH2

4-Aminobiphenyl

2-ACETYLAMINOFLUORENE (AAF)

N-HYDROXY-AAF

MULTIPLE ELECTROPHILIC METEABOLITES

NUCLEIC ACID – AND PROTEIN – BOUND AAF – AND AF - RESIDUES

COCH3

COCH3

Metabolic Activation of 2-AAF

N-Nitroso and Related Chemicals CH3–N–CH3

NO

Dimethylnitrosamine

or N-nitrosodimethyl-

amine

R–N–R1

N O Dialkyl

nitrosamine N

N

NO N-nitrosonor

-nicotine

CH3NC–NHNO2

NO N-Methyl-N1-nitro-

N- nitrosoguanidine

NH CH3 NH NH CH3 Dimethylhydrazine

CH3NCNH2

NO N-Methyl-N-nitroso-

urea

O

Nitrate Nitrite

Secondary Amine

Nitrosamine H+

Endogenous Formation of N-Nitrosamines

Macronutrients and Cancer • Fat (obesity)–colon, breast, pancreas, prostate,

endometrium, kidney, esophagus (adenocarcinomas)

• Fruits and vegetables

• Meat–colon

• Salt–stomach

Ethanol and Cancer • Liver

• Esophagus

• Oral Cavity and Pharynx

• Breast

• Colon (beer)

• Larynx

High Dose Only

Cigarette Smoking Approximately one-third of cancer deaths in United States

Lung

Larynx

Oral cavity & pharynx

Paranasal sinuses

Esophagus

Stomach

Urinary bladder

Ovary (mucinous tumors)

Pancreas

Liver

Kidney

Ureter

Uterine cervix

Bone marrow (leukemia)

Colorectal

Salivary gland (Warthin’s tumor)

Normal Processing of α2u‒Globulin Tubular Lumen

Blood

Proximal (P2) Tubule Cell

Golgi apparatus

SL SL

Degradation products

EV

EV

L SL

Blood Proximal (P2) Tubule Cell

Tubular Lumen Alpha –globulin2u + C: GA

hyaline droplets

secondary lysosome

lysosome

endocytic vesicle

Processing of Chemical Bound α2u‒Globulin

Incidence of Bladder Carcinoma in Mice Implanted with Paraffin Wax Pellets

Time (wks) Incidence (%)

40-50 70-80

100-110

10.6 26.7 53.8

Substances Producing Urinary Calculi Uracil Melamine Uric Acid Homocysteine Cysteine Calcium oxalate Calcium phosphate Diethylene glycol Biphenyl HIV Protease inhibitors PPARγ agonists

4-Ethylsulfonylnaphthalene-1-sulfonamide Oxamide Acetazolamide Terephthalic acid Dimethyl terephthalate Nitrilotriacetate Polyoxyethylene-8-stearate Glycine Orotic acid Sodium saccharin

Newer Alternatives to Carcinogenicity Testing • P53 +/- Transgenic Model (6 months)

‒ Only used to address genotoxicity • TG.AC Transgenic Model

‒ No longer used • Neonatal Mouse Model (1 year) • TGHras2 Model (6 months) • XPA-1- Repair Deficient Model (9 months)

‒ Combined with p53 +/- • Ito Medium Term Rat Assay

‒ Complex regimen

Do not address human carcinogenicity!

Lawyers As A Test Species Pros: - Plenty of Them - Will do anything, some things even a rat won’t do

- No problem with animal rights activists

- Multiply rapidly

- Don’t become emotionally attached to them

Cons: - Expensive

- Still left with interspecies extrapolation problem

Genotoxicity Screens

Induce enzymes in animal with inducer

Kill animal and remover organs

(liver, kidney, lungs)

Cofactors and Salts

Supernatant (S-9)

Liver homogenized

S-9 Mix

his- Salmonella grown in histidine- containing medium

One

Week

Centrifuge

10β Bacteria Test Chemical

Soft Agar and trace of histidine of 450 C

S-9 Mix

Transfer of mixture to petri dish Two days

incubation

at 370 C

Revertant (his+) colonies counted

Hard Agar

Structure Activity Relationships (SAR)

Halogenated methanes C(X)

X = H,F,CI, Br,1 In any combination

Chemical

Short term in vivo assay at MTD to identify possible target tissues. Possible

human carcinogen; requires risk assessment

Immunosuppressive Estrogenic activity

Yes Possible human carcinogen; requires

risk assessment

Specific evaluation to determine MOA and dose response in tissues positive

in screen

MOA and dose relevant to humans

Yes

Yes No DNA Reactive

Yes Possible human carcinogen; requires risk

assessment

Unlikely human carcinogen for intended use and expected exposure

Screening for Carcinogenesis

13 week bioassay screen to evaluate

cytotoxicity and/or cell proliferation

No

MODELS: ALL ARE WRONG, SOME ARE USEFUL.

GEORGE BOX

Basic Assumptions of Use of Bioassays for Human Risk Assessment

1.Carcinogenic effects at high doses will also occur at low doses (dose extrapolation).

2.Chemicals that cause cancer in rodents will cause cancer in humans (species extrapolation)

UNDERSTANDING COMPUTER TECHNOLOGY

PBPK Modeling

Km, Vmax, CVL

CVL

CVR

CVS

CVF

QC, CV QC, CA

QF

QS

QR

QL

KME, AM

Urine (Metabolite)

Metabolite

LUNG

Organs

Muscle

Fat

Liver

Veno

us B

lood

IV Injection Drinking Water Gavage

Inhalation QP, CI QP, CX

ED01 Megamouse Experiment (2-AAF)

Liver Bladder

Dose (mg/kg Diet)

fmol

Add

uct p

er u

g D

NA

AAF-DNA Adducts

AAF-Induced Bladder Hyperplasia

Synergies Between Genotoxic and Proliferating Agents

Cigarette Smoke Hepatitis Virus & Aflatoxin Papilloma Virus & Cigarette Smoke Helicobacter & N-Nitroso Compounds

Lung, Bladder Liver Cervix Stomach

Factors Influencing Carcinogenesis

The Human Relevance Framework 1. Is the weight of evidence sufficient to establish the MOA in animals?

a. Postulated MOA b. Identification of key events c. Animal evidence d. Application of DPA/IPCS animal MOA guidance (Table 2)

2. Are key events in the animal MOA plausible in humans?

a. Concordance analysis of animal and human responses b. Statement of confidence

3. Taking into account kinetic and dynamic factors, is the animal MOA plausible in humans?

1. Concordance analysis of animal and human responses 2. Statement of confidence

4. Statement of confidence; analysis; implications

Crit. Rev. Toxicol., 33: 593, 2003

Overview/Objectives 1. Principles of Carcinogenesis 2. DNA Reactive and Non-DNA Reactive Carcinogens 3. Methods for Screening for Carcinogenicity 4. Model of Carcinogenesis Incorporating DNA Damage

and Increased Cell Proliferation 5. Mode of Action Analysis and Extrapolation of In Vitro

and Animal Studies to Humans

References 1. Cohen, SM. Evaluation of Possible Carcinogenic Risk to Humans Based on Liver

Tumors in Rodent Assays: The Two-Year Bioassay is No Longer Necessary. Toxicol. Pathol., 38: 487-501, 2010.

2. Cohen, SM and Arnold, LL. Chemical Carcinogenesis. Toxciol. Sci., 12 (Suppl. 1): 576-592, 2011.

3. Cohen, SM and Ellwein, LB. Cell Proliferation in Carcinogenesis. Science, 249: 1007-1011, 1990.

4. Cohen, SM and Ellwein, LB. Genetic Errors, Cell Proliferation and Carcinogenesis. Cancer Res., 51: 6493-6505, 1991.

5. Boobis, AR, et al. IPCS Framework for Analyzing the Relevance of a Cancer Mode of Action for Humans. Crit. Rev. Toxicol., 36: 781-792, 2006.

6. Embry, MR, et al. Risk Assessment in the 21st Century: Roadmap and Matrix. Crit. Rev. Toxicol., 44 (Suppl. 3): 6-16, 2014.

Chemical Carcinogenesis Samuel M. Cohen, MD, PhD

University of Nebraska Medical Center Department of Pathology and Microbiology

Omaha, NE